Tumors with mutant BRAF or mutant KRAS are dependent on ERK signaling and are sensitive to inhibitors of the pathway. Selective RAF, MEK and ERK inhibitor have now been developed as therapeutics for these tumors, however, their clinical efficacy is limited by toxicity, acquired resistance, and adaptive resistance due to relie of ERK-dependent feedback inhibition of mitogenic signaling. Our previous work shows that whereas MEK and ERK inhibitors suppress ERK signaling in all normal and tumor cells, RAF inhibitors only inhibit signaling in tumors with BRAF mutations and activate ERK signaling in other tissue. Thus, RAF inhibitors have a broader therapeutic index than MEK and ERK inhibitors, but only work in tumors with mutant BRAF. We have also shown that inhibition of ERK signaling by all of these inhibitors reactivates feedback inhibited signaling via RAF/ERK and non-RAF/ERK pathways and that this feedback activation limits the antitumor effects of RAF and MEK inhibitors. Our goals in this proposal are to develop therapies that maximally inhibit ERK output by combining RAF or MEK inhibitors with selective novel MEK and ERK inhibitors that suppress feedback reactivation of ERK signaling. Our data suggests that this is required for maximal antitumor activity, but that it will also relieve feedback inhibition of RTK activation resulting in potent activation of other signaling pathways that can attenuate efficacy. We plan to identify these reactivated pathways and then develop and test therapies that combine maximal ERK inhibition with inhibition of key reactivated pathways to prevent or limit adaptive resistance. Specific regimens will be designed for initial treatment of mutant BRAF tumors (which will employ RAF inhibitors) and for tumors with acquired resistance to RAF inhibitors or with RAS mutation (which will not). While preclinical models are powerful tools, ultimately mechanisms of resistance need to be identified and validated in clinical specimens from melanoma patients treated with these agents. We will thus use targeted DNA and RNA-sequencing methods to define the basis for RAF-inhibitor resistance using tumor samples collected pre-treatment and at the time of disease progression on RAF inhibitors. One goal of these latter studies will be to determine whether we can predict, prior to drug treatment, the mechanism of RAF-inhibitor resistance with the goal of using the data to develop individualized treatment strategies that delay or prevent the emergence of drug resistant clones.